The present invention relates to a competitive binding assay kit and process for the immunological detection of a presence of a blood antigen of specified phenotype (for example phenotypes of HPA) in a sample of whole blood.
Human Platelet Antigen 1 (HPA1), also known as PLA1, is a polymorphic determinant on the glycoprotein (GP) IIb/IIIa complex, that resides at the N-terminal region of GPIIIa. The great majority of the Caucasian population is homozygous or heterozygous for HPA1a (about 72% and 26% respectively) (1). However, 2-3% of the population is homozygous for HPA1b (PLA1 negative) putting them at risk from developing antibodies to HPA1a either during pregnancy or following transfusion with blood containing HPA1a platelets. For example, HPA1b pregnant women carrying HPA1a positive babies may produce anti-HPA1a antibodies which could lead to fetal or neonatal alloimmune thrombocytopenia (FAITP or NAITP) respectively, with 50% of the cases occurring at first pregnancy. One outcome of FAITP/NAITP, which has a frequency of 1 in 1000-2000 births, is intracranial hemorrhage with 6.5-10% mortality and 20% morbidity (neurological sequelae) (2).
Moreover, HPA1b individuals (mostly elderly multi-parous women) transfused with blood containing HPA1a platelets may develop post-transfusion purpura (PTP) which is characterized with severe thrombocytopenia, 5-10 days following the transfusion. Although not as common as NAITP (only about 200 cases reported world-wide), PTP is nevertheless associated with 10-20% morbidity/mortality (3).
The treatment and management of FAITP/NAITP and PTP relies basically on several infusions of intra-venous IgG with or without transfusion with HPA1b platelets. In view of the serious or fatal outcomes of FAITP/NAITP and PTP and their increasingly effective treatment, antenatal screening for HPA1b is becoming widely accepted as an important prophylactic measure (4,5). For such wide-scale screening, established assays such as monoclonal antibody immobilization of platelet antigens (MAIPA), platelet suspension immunofluorescence test, or flow cytometry are not suitable due to their technical or financial burdens (6-8). Recently, Metcalfe et al reported a simplified method for large-scale HPA1a phenotyping for antenatal screening which was adapted from MAIPA (9). However, the Metcalfe et al assay relies on the extraction of glycoprotein GPIIb/IIIa antibody complex from platelets pre-incubated with the antibody. The assay is also considerably time consuming requiring a number of incubation and centrifugation steps.
EP 0028133 describes a method of detecting and quantitating haptens and antigens. A competitve binding assay is described in which a sample and a reagent containing an immunoreactive antibody are in a liquid phase, the sample and reagent either being mixed together or reacted before contacting a solid phase onto which a known concentration of a substance selected from haptens, antigens and/or mixtures thereof, which are also reactive with said antibody, is bound.
There is thus a requirement for an immunoassay which does not require a sample of whole blood to be substantially purified.
It is an object of the present invention to obviate and/or mitigate some of the above disadvantages.
Generally speaking the present invention provides a simple and potentially automatable competitive binding assay and assay kit for determining the presence in vitro of a specified blood antigen (for instance HPA1a) in a sample of whole blood. It is generally based on the surprising feature that the assay is able to determine a presence or absence of a specified blood antigen in a sample of whole blood. It might have been expected that cells and other species normally present in whole blood would interfere with such an assay.
The present invention provides in a first aspect a method of performing a competitive binding immunoassay for determining in vitro the presence of a specified blood antigen phenotype in a sample of whole blood, which comprises:
mixing the sample of whole blood with a predetermined amount of antibody specific to said blood antigen phenotype, so as to bind the antibody to any of said antigen present in the sample of whole blood;
bringing the mixture into contact with a substrate upon which antigen of said phenotype has been immobilized such as to bind any remaining unbound antibody to said immobilized antigen, thereby forming an immobilized antigen-antibody complex;
washing the immobilized antigen-antibody complex and;
estimating the amount of immobilized complex and consequently the specified blood antigen phenotype.
The present invention in a further aspect provides a competitive binding immunoassay kit for determining the presence of a specified blood antigen phenotype in a sample of whole blood which, comprises;
a substrate having immobilized thereon substantially pure blood antigen of said phenotype.
a supply of antibody for specifically binding to said antigen, and
means for enabling detection of said specifically bound antibody.
The immunoassay of the present invention is described as a competitive binding immunoassay essentially because any blood antigen of said specified phenotype present in the whole blood sample competes with said immobilized antigen, for the predetermined amount of antibody.
The immunoassay can be used to detect the presence of any suitable intrinsic blood cell antigen of a specified phenotype (excluding foreign species such as viral antigens etc.). The blood antigens include antigenic determinants on platelet glycoproteins, in particular HPA1 antigen of 1a or 1b phenotype. Other antigens which are also suitable are glycoproteins or other determinants on blood cells, particularly white blood cells (such as granulocytes).
The sample of whole blood may be obtained fresh from an individual to be tested, or the blood may be from whole blood stored up to 25 days at 4xc2x0 C. (for example citrated or EDTA whole blood). The whole blood sample may be used untreated, or alternatively the whole blood sample can be subjected to a washing step prior to testing (e.g. by diluting the blood, centrifuging it and resuspending in a buffer).
Typically, a sample of serum containing a high titer of antibody specific to said blood antigen may be employed in the immunoassay. Alternatively a monoclonal antibody specific to said blood antigen could be used.
The substrate upon which said substantially pure antigen of said phenotype is immobilized, can be any suitable substrate known in the art. Typically this can be paper, plastics such as nitrocellulose, or glass (e.g. microscope slides). Most preferably the substrate is a well of a plastics microtiter plate, as commonly employed in immunoassays.
The antigen to be immobilized is preferably substantially pure. That is, the antigen is purified away from other blood antigens or blood components that could affect the immunoassay. It is important that the antigen be substantially pure if the antibody used is in a serum sample. This is to prevent any additional antibodies that may be present in the serum sample, from binding to their respective blood antigen. If a monoclonal antibody is employed, it may not be necessary to highly purify the antigen, depending on the degree of cross-reactivity of the monoclonal antibody which should be specific to its specified antigen. The antigen used should be purified from a blood source, different to that being tested. This is to minimize any further antigen-antibody complexes which may be formed, from interfering with the immunoassay.
Typically a number of immune (i.e. antigen-antibody) complexes may be formed when bringing the mixture into contact with the substrate, for instance; a) specified antibody and immobilized antigen, b) specified antibody and antigen present in the whole blood, and c) non-specifically bound unspecified antibody to immobilized antigen. The washing serves to remove all complexes except the specified antibody and immobilized antigen complex.
The mixing of the sample of whole blood with antibody and the bringing the mixture into contact with the substrate can be carried out concurrently, or alternatively as two separate steps.
Generally, the substrate bound immune complex is estimated by means of anti-human IgG or IgM labelled with an appropriate label, such as an enzyme label (e.g. horseradish peroxidase HRP), a radiolabel, a fluorescence label or other labelling system known in the art. Using an enzyme label, an enzyme-linked immunosorbent assay (ELISA) may be provided which is sensitive, specific, cheap, quick and could potentially be used for large scale automated screening for whole blood antigens. Advantageously, it has been found that the incubations involved in the production of the immune complex, and the estimation thereof with labelled antibody can be carried out at room temperature and so do not require the use of an incubator.
It is envisaged that a particularly preferred immunoassay would involve detection of HPA1a antigen. The antigenic determinant for HPA1a is present on the GPIIIa subunit, so that a competitive binding immunoassay to detect the presence HPA1a antigen in a whole blood sample, would include immobilized HPA1a antigen. Typically this can be HPA1a typed platelets (which have been previously genotyped, for instance by using the technique described in Williamson, et al (13)), purified glycoprotein complex GPIIb/IIIa (prepared, for example, by the process described in Bessos et al (10)), purified glycoprotein GPIIIa, or synthetically manufactured HPA1a antigen.
In a particularly preferred embodiment of the first aspect, the present invention provides a method of performing a competitive binding immunoassay for determining in vitro the presence of HPA1a antigen in a sample of whole blood, which comprises;
mixing the sample of whole blood with a predetermined amount of anti-HPA1a antibody, so as to bind the antibody to any HPA1a antigen present in the sample of whole blood;
bringing the mixture into contact with a substrate upon which HPA1a antigen has been immobilized, such as to bind remaining anti-HPA1a antibody to the immobilized HPA1a antigen, thereby forming an immobilized HPA1a antigenxe2x80x94anti-HPA1a antibody complex;
washing the immobilized HPA1a antigenxe2x80x94anti-HPA1a antibody complex; and
estimating the amount of immobilized complex and consequently HPA1a antigen.
In a particularly preferred embodiment of the further aspect, the present invention provides a competitive binding immunoassay kit for determining the presence of HPA1a antigen in a sample of whole blood which comprises;
a multiwell microtiter plate or plates having a well containing immobilized substantially pure HPA1a antigen;
a supply of anti-HPA1a antibody for specifically binding to said HPA1a antigen, and
means for enabling detection of said specifically bound antibody.
Typically, each well contains 0.05 xcexcg to 0.5 xcexcg of antigen per well; preferably 0.1 xcexcg of antigen per well.
The substrate upon which HPA1a antigen has been immobilized is generally blocked with a non-specific protein mixture, such as bovine serum albumin prior to bringing the mixture into contact with the substrate.